This invention relates to a method of characterizing polynucleotides.
It is often necessary to characterize polynucleotides for quality control or other purposes. This is particularly necessary when polynucleotides are manufactured and some degree of consistency must be maintained between batches and the overall characteristics of the polynucleotide must be ascertained simply to verify that the product produced is that desired. The manufacture of polynucleotides is well known. For example, techniques for the manufacture of ssRNA are described in Carter, W. A., Pitha, P. M., Marshall, L. W., Tazawa, I., Tazawa, S., and Ts'o, P. O., (1972) J. Mol. Biol. 70, 567-587; Carter, W. A., O'Malley, J. A., Beeson, M., Cunnington, P., Kelvin, A., VereHodge, A., Alderfer, J. L., and Ts'o, P. O. (1976) Molecular Pharmacology 12, 440-453; Ts'o, P. O. P., Alderfer, J. L., Levy, J., Marshall, L. W., O'Malley, J. A., Horoszewicz, J. S., and Carter, W. A. (1976) Molecular Pharmacology 12, 299-312; and Greene, J. J., Alderfer, J. L., Tazawa, I., Tazawa, S., Ts'o, P. O., O'Malley, J. A., Carter, W. A. (1978) Biochemistry 17, 4214-4220.
Current techniques available to analyze polymers included gel electrophoresis and size exclusion chromatography (SEC). Size exclusion chromatography, although commonly used in typical polymer analysis, was limited in its inability to retain molecules beyond 1,000,000 molecular weight, and this was unsuitable for the polynucleotide application. A further limitation of this method involves the shearing of large molecules when forced at high pressure through column packings of limited pore size. Consequently, erroneous sizing of molecules occurs in the size distribution from molecular breakdown.
Gel electrophoresis, using agarose as the gel matrix has proved to be an extremely suitable technique for characterizing the entire size distribution range of biopolymers having high molecular weights. The concentration of the gel matrix was easily adjusted to accommodate the extensive range of size distribution. After electrophoresis, the gels were scanned by laser densitometry, however, no method existed to convert the densitometry data into size distribution parameters. Current software generally is limited to quantitation of discreet bands of nucleic acids and not size distribution type analyses.
Hence, current techniques have not been entirely satisfactory for the purpose of characterizing polydispersed polynucleotide size distributions.
Freeman, et al. in their article appearing in Analytical Biochemistry 158, 119-129 (1986) describe a technique for characterizing DNA by quantitating the molecular size distribution by determining strand breaks in deoxyribonucleic acid (DNA). Freeman, et al. disclose only a very rudimentary system involving determining number average molecular lengths and length average molecular lengths of DNA by using rather the mobilities of the molecular length standards. This is too rudimentary and crude to provide a valuable analysis tool.
Kohen et al., Analytical Biochemistry, 154, 485-491 (1986) describe a technique for determining number average molecular weight by measuring absorbance to ascertain single strand breaks. Kohen et al. apparently do not determine weight average molecular weight, Z average molecular size, polydispersity or skewness.
Bindels et al. in Journal of Chromatography, 252; 255-267 (1982) describe a technique of using high performance gel permeation chromatography to determine the weight average and number average molecular weights for certain materials. A disadvantage of such a chromatographic technique has been discussed above.